Bacterial and fungal infections are major causes of morbidity and mortality, especially in newborns and immunocompromised patients. Rapid diagnosis of these infections would allow for the early institution of appropriate antimicrobial therapy. Currently available cultivation techniques and antigen detection assays are often not sufficiently rapid to provide the information needed to institute effective therapy. However, the rapid direct measurement of specific bacterial or fungal enzymes which do not occur in unifected human cells or body fluids would provide a means for rapid diagnosis of microbial infections. Three enzymes which occur in a wide range of bacterial and fungal organisms but not in mammalian cells are Beta-lactamase, adenine permease and adenine aminohydrolase. The presence of one or more of these microbial organisms in the blood or normally sterile body fluid of a person would thus be indicative of the presence of an infecting microorganism. In preliminary studies we have developed an enzymatic radioisotopeassay (ERIA) which is approximately 100 fold more sensitive than previously described assay for the measurement of Beta-lactamase. When applied to the measurement of Beta-lactamase in clinical specimens the ERIA detected Beta-lactamase in the blood, cerebrospinal fluid, pleural fluid and joint fluid of 59 of 62 patients with documented infections caused by Beta-lactamase producing organisms. Similarly, we have developed sensitive ERIA's which are capable of measuring small amounts of adenine permease and adenine aminohydrolase, enzymes found exclusively in bacterial and fungal organisms. These assays were also capable of detecting small quantities of infecting bacterial and fungal organisms. We plan to adapt these assays so that they can provide information as to the antibiotic sensitivity of the infecting microorganism. In addition, we propose to adapt these assays to non radiometric methods so that they can be utilized in a large number of different clinical settings. The availability of simple, rapid diagnostic tests for microbial infection would markedly improve the health care of patients with suspected infectious diseases.